Preliminary evidence suggests that the process of melanoma tumorigenesis is promoted by aberrant constitutive production of the inducible form of nitric oxide synthase (iNOS), an enzyme responsible for production of nitric oxide (NO). The iNOS protein was first recognized as expressed by immune system cells in response to cytokine stimulation, generating muM levels of NO which directly causes the death of certain bacteria as well as local tissue damage. In contrast low (nM-pM) levels of NO production by human tumors have been proposed as responsible for increased growth, invasion, angiogenesis, genomic instability, and resistance to apoptosis as reported in leukemia, breast, colon, pancreas, and brain tumors. Our preliminary analysis of iNOS in human melanoma tumor specimens from advanced (Stage III) patients indicates that the 60% of patients whose tumors express iNOS are more likely to die within 20 months of therapy, than the 40% of patients with undetectable iNOS (p <0.0010). The functional consequences and molecular mechanisms that occur as a result of iNOS expression are critical to resolve, and are directly tested for human melanoma in the four aims of this proposal. Aim I studies the effect of iNOS levels in melanoma cell lines and whether NO levels are related to growth, vascular mimicry, invasion, or apoptosis resistance; a series of iNOS and NO inhibitors are also tested here for effectiveness in reversing the hypothesized effects. Aim II address molecular mechanisms of NO in melanoma, with an initial emphasis on the nitration of tumor suppressor proteins. Preliminary evidence suggests that a functional inactivation of wtp53 may be directly related to nitration of the p53 molecule in iNOS positive tumors. Aim III develops an in vivo model of human melanoma testing aggressiveness of regulatable iNOS in a SCID mouse model, and to apply inhibitors found in Aim I as potential therapeutic agents. Finally, Aim IV will determine if 1NOS protein presence in patient's tumors continues to predict those patients with worse prognosis. Studies of iNOS regulation and NO inhibitors may provide insight into novel therapeutic approaches, if it is found that reduction or elimination of iNOS reduces tumor growth and restores sensitivity to apoptosis and therapeutic agents. Therefore, the analysis of INOS and the functional, molecular, and prognostic consequences of iNOS expression are proposed for critical evaluation in this application.